Nuclear hormone receptor fluorescence polarization assay

ABSTRACT

Methods for identifying modulators of nuclear hormone receptor function comprise the steps of (a) forming a mixture comprising a nuclear hormone receptor, a peptide sensor and a candidate agent, but not a natural coactivator protein of the receptor, wherein the sensor provides direct, in vitro binding to the receptor under assay conditions; (b) measuring an agent-biased binding of the sensor to the receptor; and (c) comparing the agent-biased binding with a corresponding unbiased binding of the sensor to the receptor. In particular embodiments, the sensor comprises an amphipathic alpha helix nuclear hormone interacting domain comprising a recited nuclear hormone transcriptional coactivator motif sequence, the sensor is present at sub-micromolar concentration, the binding reaction occurs in solution, the sensor comprises a fluorescent label and the measuring step comprises detecting fluorescence polarization of the label. Reagents include labeled sensor peptides and reaction mixtures consisting essentially of nuclear hormone receptor, a peptide and a candidate agent.

FIELD OF THE INVENTION

The field of this invention is screens for drugs effecting nuclearhormone receptor function.

BACKGROUND

Nuclear hormone receptors comprise a large, well-defined family ofligand-activated transcription factors which modify the expression oftarget genes by binding to specific cis-acting sequences (Laudet et al.,1992, EMBO J, Vol, 1003-1013; Lopes da Silva et al., 1995, TINS 18,542-548; Mangelsdorfet al., 1995, Cell 83, 835-839; Mangelsdorf et al.,1995, Cell 83, 841-850). Family members include both orphan receptorsand receptors for a wide variety of clinically significant ligandsincluding steroids, vitamin D, thyroid hormones, retinoic acid, etc.Ligand binding is believed to induce a conformational change in thereceptors and promote their association with transcriptionalcoactivators, which are a diverse group of large nuclear proteins (Glasset al., 1997, Curr Opn Cell Biol 9, 222-232), which may share asignature sequence motif (Heery et al., 1997, Nature 733-736). Theresulting complex then binds high affinity sites in chromatin andmodulates gene transcription.

The classic approach to identifying agonists or antagonists of nuclearhormone receptors is the ligand displacement assay, where thedisplacement of radiolabeled ligand by candidate agents is detected. Analternative approach is a cell-based transcription assay for expressionof a reporter of nuclear hormone receptor activation (e.g. Evans et al.(1991) U.S. Pat. No. 5,071,773). More recently, a gel-based coactivatordependent receptor ligand assay (Krey et al., 1997, Mol Endocrinol 11,779-791) has been used to identify ligands of peroxisomeproliferator-activated receptors (PPARs), which are nuclear hormonereceptors activated by a variety of compounds including hypolipidemicdrugs. Unfortunately, these various assays suffer from a number oflimitations including a required known ligand and time, labor andresource intensive cell-based and gel-based methods, respectively.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for efficient screeningof modulators of nuclear hormone receptor function, without the use ofcell- or gel-based steps. The methods are amenable to automated,cost-effective high throughput screening of chemical libraries forbioactive compounds.

In one embodiment, the invention provides in vitro methods comprisingthe steps of (a) forming a mixture comprising a nuclear hormonereceptor, a peptide sensor and a candidate agent, but not a naturalcoactivator protein of the receptor, wherein the sensor provides direct,in vitro binding to the receptor under assay conditions; (b) measuringan agent-biased binding of the sensor to the receptor; and (c) comparingthe agent-biased binding with a corresponding unbiased binding of thesensor to the receptor, wherein a difference between the biased andunbiased bindings indicates that the agent modulates a receptorfunction. In particular embodiments, the sensor comprises an amphipathicalpha helix nuclear hormone interacting domain comprising a nuclearhormone transcriptional coactivator motif sequence. To ensurespecificity and optimize binding, the sensor is generally present atsub-micromolar concentration and the binding reaction occurs insolution. In a preferred embodiment, the sensor comprises a fluorescentlabel and the measuring step comprises detecting fluorescencepolarization of the label.

The invention also provides reagents such as labeled sensor peptides andreaction mixtures consisting essentially of nuclear hormone receptor, apeptide and a candidate agent, wherein the peptide provides direct, invitro ligand-dependent binding to the receptor, especially in which thebinding is enhanced in the presence of the agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. LXR activation dose response with oxysterol ligands influorescent polarization assay with TUK-1391 sensor.

FIG. 2. Dose response showing 24-ketocholesterol ligand (2 μM) increasesLXR receptor affinity for labeled peptide sensor in fluorescentpolarization assay.

FIG. 3. Dose response showing 9-cis-retinoic acid ligand (1 μM)increases RXR receptor affinity for labeled peptide sensor influorescent polarization assay.

FIG. 4. Fluorescent polarization NRH agonist assay validation forLXR/24-ketocholesterol ligand (2 μM), PPARγ/BRL49653 (1 μM) andRXR/9-cis-retinoic acid ligand (1 μM) with TUK-1391 sensor.

DETAILED DESCRIPTION OF THE INVENTION

The methods generally employ a mixture comprising three components: anuclear hormone receptor, a peptide sensor and a candidate agent, inamounts effective to measure the targeted interactions. Many naturalnuclear hormone receptors are modular proteins with discrete functionaldomains, including a ligand binding domain; see Laudet et al.; Lopes daSilva et al.; Mangelsdorf et al.; supra. The subject receptors encompasssuch full-length receptors as well as portions of the receptorssufficient to provide differential sensor binding in the presence andabsence of a corresponding receptor ligand, agonist and/or antagonist.Such portions generally comprise at least the ligand binding domain ofthe receptor. A wide variety of molecular and biochemical methods areavailable for biochemical synthesis, molecular expression andpurification of the subject compositions, see e.g. Molecular Cloning, ALaboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory),Current Protocols in Molecular Biology (Eds. Ausubel, et al., GreenePubl. Assoc., Wiley-Interscience, N.Y.) or that are otherwise known inthe art. Exemplary nuclear hormone receptors and corresponding targettherapeutic application are listed in Table 1.

TABLE 1 Exemplary nuclear hormone receptors, form (M = monomeric, D =heterodimeric, H = homodimeric), tissue expression and targettherapeutic application Receptor Form Tissue Expression TargetTherapeutic Application NURR1 M/D Dopaminergic Neurons Parkinson'sDisease RZRβ M Brain (Pituitary), Muscle Sleep Disorders RORα MCerebellum, Purkinje Cells Arthritis, Cerebellar Ataxia NOR-1 M Brain,Muscle, Heart, Adrenal, Thymus CNS Disorders, Cancer Rev-ErbAβ H Brain,Muscle, Spleen CNS Disorders T1x H Embryonic and Adult Brain CNSDisorders NGFI-Bβ M/D Brain CNS Disorders HZF-2α H Hippocampus CNSDisorders COUP-TFα H Brain CNS Disorders COUP-TFβ H Brain CNS DisordersCOUP-TFγ H Brain CNS Disorders Nur77 M/D Brain, Thymus, Adrenals CNSDisorders LXRα D Liver, Kidney, Spleen, Adrenals HypercholesterolemiaCOR M Liver, Pancreas Hypercholesterolemia Rev-ErbAα H Muscle, Brain(Ubiquitous) Obesity HNF4α H Liver, Kidney, Intestine Diabetes TOR MThymus, T Cells, Lymphoma Immune Disorders MB67α D Liver MetabolicDisorders SHP D Liver, Heart, Pancreas Metabolic Disorders FXR D Liver,Kidney Metabolic Disorders SF-1 M Gonads, Pituitary Metabolic DisordersLXRβ D Kidney (Ubiquitous) Metabolic Disorders GCNF M/H Testes, OvaryInfertility TR2-11α,β H Testes Infertility, Contraception TR4 H TestesInfertility, Contraception ERRα,β M Placenta Infertility DAX-1 M Testes,Adrenals, Ovary, Liver Adrenal Hypoplasia, Hypogonadism

The mixture also includes a peptide sensor which provides direct, invitro, significant assay detectable binding to the receptor under assayconditions. Accordingly, the sensor obviates the need to include anatural coactivator protein of the receptor in the mixture. The sensorcomprises a receptor binding sequence, generally L₁X₁X₂L₂L₃, whereinL₁-L₃ are independently selected from hydrophobic amino acids,preferably leucine or isoleucine, more preferably leucine; and X₁-X₂ areindependently selected from any amino acid, preferably any natural aminoacid. The sensor region comprising this sequence generally forms anamphipathic alpha helix. Such sequences may be natural coactivatorprotein motif sequences, derived from coactivator motif sequences orconsensus sequences thereof, e.g. by step-wise mutational analysis,and/or from screens of purely or partly synthetic sequences, e.g.randomizing residues and selecting for receptor binding. The sensors areof length and sequence sufficient to effect the requisite specificbinding, generally 50 or fewer, preferably 24 or fewer, more preferably12 or fewer residues in length. Accordingly, panels of predetermined orrandomized candidate sensors are readily screened for receptor binding.For example, in the high-throughput fluorescent polarization assay(below), candidate sensors demonstrating specific binding areconveniently identified by enhanced fluorescent polarization, generallyan increase of at least about 5, preferably at least about 10, morepreferably at least about 20 millipolarization units under optimizedbinding assay conditions.

In a particular embodiment, the sensors demonstrate ligand, agonistand/or ligand dependent binding i.e. the sensor differentially binds thereceptor in the presence and absence of such ligand/agonist/antagonist,generally differential binding of at least 10/10%, preferably at least100/50%, more preferably at least 1,000/90%, respectively. Accordingly,panels of predetermined or randomized candidate sensors are readilyscreened for differential binding, as exemplified in FIGS. 2 and 3 fortwo exemplary receptor/ligand pairs. Analogously, differential bindingis conveniently demonstrated using known agonists or antagonists oftargeted receptors. For orphan receptors, it is often convenient toprescreen known ligands for pseudo-ligands or surrogates whichselectively bind the ligand binding domain. Alternatively, agonistsand/or antagonists may be identified by screening predetermined orrandomized candidate labeled peptides for sensors which demonstrateassay detectable receptor binding, and then screening for agents whichincrease/decrease the binding of the identified sensor to the receptor,i.e. agonists/antagonists, respectively.

Exemplary sensors and binding data are shown in Table 2.

TABLE 2 Sensors Activity: Fluorescent Polarization Assay Sensor LabelSequence LXR PPARγ RXR SRC-1 632-640 TUK-1384 F- KLVQLLTTT SEQ ID NO: 1I O O TUK-1386 F-G- KLVQLLTTT I O O TUK-1385 R- KLVQLLTTT II+ I II+TUK-1387 R-G- KLVQLLTTT + O II SRC-1 689-696 TUK-1370 F- ILHRLLQE II OII TUK-1371 R- ILHRLLQE SEQ ID NO: 2 IV I+ IV TUK-1373 R-G- ILHRLLQE IIO II+ SRC-1 748-755 TUK-1390 F- LLRYLLDK SEQ ID NO: 3 IV II II+ TUK-1392F-G- LLRYLLDK II+ I I TUK-1391 R- LLRYLLDK IV II+ IV TUK-1393 R-G-LLRYLLDK III+ I II+ SRC-1 748-754 TUK-1453 R- LLRYLLD SEQ ID NO: 4 III II+ SRC-1 749-754 TUK-1455 R- LRYLLD SEQ ID NO: 5 IV I+ I SRC-1 748-753TUK-1457 R- LLRYLL SEQ ID NO: 6 II + I SRC-1 749-753 TUK-1459 R- LRYLLSEQ ID NO: 7 III I O SRC-1 748-756 TUK-1472 R- LLRYLLDKD SEQ ID NO: 8 IVII IV SRC-1 747-756 TUK-1473 R- QLLRYLLDKD SEQ ID NO: 9 IV I+ I+ SRC-1746-756 TUK-1474 R- HQLLRYLLDKD SEQ ID NO: 10 IV O I SRC-1 1427-1440TUK-1395 F- PQAQQKSLLQQLLT SEQ ID NO: 11 O O O TUK-1397 F-G-PQAQQKSLLQQLLT O O O TUK-1398 R-G- PQAQQKSLLQQLLT O O O SRC-1 1434-1441TUK-1380 F- LLQQLLTE SEQ ID NO: 12 II+ O O TUK-1382 F-G- LLQQLLTE II+ OO TUK-1381 R- LLQQLLTE IV I+ I+ TUK-1383 R-G- LLQQLLTE II O O RIP-140496-506 TUK-1374 F- VTLLQLLLG IV I O TUK-1376 F-G- VTLLQLLLG SEQ ID NO:13 II+ + O TUK-1375(1433) R- VTLLQLLLG IV I O TUK-1377 R-G- VTLLQLLLG III + Synthetic sequence peptides TUK-1560 R- ILRKLLQE SEQ ID NO: 14 IV IIIV TUK-1559 R- ILKRLLQE SEQ ID NO: 15 IV 0 IV TUK-1558 R- ILRRLLQE SEQID NO: 16 III 0 IV TUK-1557 R- ILKKLLQE SEQ ID NO: 17 III+ 0 IV

The sensor also comprises a detectable label. A wide variety of labelsmay be used including labels providing for direct detection such asradioactivity, luminescence, optical or electron density, etc. orindirect detection such as an epitope tag, etc. A variety of methods maybe used to detect the label depending on the nature of the label andother assay components, e.g. through optical or electron density,radiative emissions, nonradiative energy transfers, etc. or indirectlydetected with antibody conjugates, etc. In a particular embodiment, thelabel is differentially detectable according to receptor binding,obviating the need for any bound versus unbound separation step. In amore particular embodiment, the label is a fluorescent label whichprovides differential fluorescence polarization depending on receptorbinding. Exemplary such labels include rhodamine and fluorescein, whichmay be coupled directly or indirectly though a linker, e.g. an aminoacid linker. Suitable labels and methods for peptideconjugation/incorporation (e.g. during solid phase peptide synthesis)are well known in the art. The sensor is generally present at aconcentration of less than about 1 μM, preferably less than about 100nM, more preferably less than about 10 nM and most preferably less thanabout 1 nM.

The assay mixture also comprises a candidate agent. Suitable candidateagents encompass numerous chemical classes, though typically they areorganic compounds; preferably small organic compounds and are obtainedfrom a wide variety of sources including libraries of synthetic ornatural compounds. In a particular embodiment, the assay mixture alsocomprises a known ligand of the receptor. This embodiment isparticularly suitable for screening for antagonists of the receptor. Avariety of other reagents may also be included in the mixture. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, protease inhibitors, etc. may be used.

The mixture is incubated under conditions whereby, but for the presenceof the candidate agent, the sensor binds the receptor with a referencebinding affinity. In a particular embodiment, all the components of themixture, including the receptor, peptide and agent, are in solution. Themixture components can be added in any order that provides for therequisite bindings and incubations may be performed at any temperaturewhich facilitates optimal binding. Incubation periods are likewiseselected for optimal binding but also minimized to facilitate rapid,high-throughput screening. After incubation, the agent-biased bindingbetween the sensor and receptor is detected according to the nature ofthe label, as described above. A difference in the binding in thepresence and absence of the agent indicates that the agent modulates areceptor binding function. A difference, as used herein, isstatistically significant and preferably represents at least a 50%, morepreferably at least a 90% difference.

The invention also provides reagents for use in the subject methods. Forexample, the invention provides sensors consisting of, or consistingessentially of, a peptide comprising the sequence L₁X₁X₂L₂L₃ SEQ ID NO:18 covalently coupled to a detectable label, wherein L₁-L₃ areindependently selected from hydrophobic amino acids and X₁-X₂ areindependently selected from any amino acid and wherein the peptideprovides direct, in vitro ligand-dependent binding to a nuclear hormonereceptor. In a particular embodiment, the label is a fluorescent labelcoupled to the N-terminus of the peptide and the peptide is 24,preferably 18, more preferably 12, most preferably 8 or fewer residuesin length. The invention also provides reagent mixtures, such as amixture consisting essentially of nuclear hormone receptor, a peptideand a candidate agent, wherein the peptide provides direct, in vitroligand-dependent binding to the receptor, preferably wherein the bindingis enhanced in the presence of the agent.

The following example is offered by way of illustration and not by wayof limitation.

EXAMPLE I. High-Throughput In Vitro Fluorescence Polarization Assay

Reagents:

Sensor: Rhodamine-labeled L₁X₁X₂L₂L₃ peptide (final conc.=1-5 nM)

Receptor: Glutathione-S-transferase/nuclear hormone receptor ligandbinding domain fusion protein (final conc.=100-200 nM)

Buffer: 10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6

Protocol:

1. Add 90 microliters of peptide/NHR mixture to each well of a 96-wellmicrotiter plate.

2. Add 10 microliters of test compound per well.

3. Shake 5 min and within 5 minutes determine amount of fluorescencepolarization by using a Fluorolite FPM-2 Fluorescence PolarizationMicrotiter System (Dynatech Laboratories, Inc).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

18 1 9 PRT Artificial Sequence Description of Artificial Sequence NHRSensor Peptides 1 Lys Leu Val Gln Leu Leu Thr Thr Thr 1 5 2 8 PRTArtificial Sequence Description of Artificial Sequence NHR SensorPeptides 2 Ile Leu His Arg Leu Leu Gln Glu 1 5 3 8 PRT ArtificialSequence Description of Artificial Sequence NHR Sensor Peptides 3 LeuLeu Arg Tyr Leu Leu Asp Lys 1 5 4 7 PRT Artificial Sequence Descriptionof Artificial Sequence NHR Sensor Peptides 4 Leu Leu Arg Tyr Leu Leu Asp1 5 5 6 PRT Artificial Sequence Description of Artificial Sequence NHRSensor Peptides 5 Leu Arg Tyr Leu Leu Asp 1 5 6 6 PRT ArtificialSequence Description of Artificial Sequence NHR Sensor Peptides 6 LeuLeu Arg Tyr Leu Leu 1 5 7 5 PRT Artificial Sequence Description ofArtificial Sequence NHR Sensor Peptides 7 Leu Arg Tyr Leu Leu 1 5 8 9PRT Artificial Sequence Description of Artificial Sequence NHR SensorPeptides 8 Leu Leu Arg Tyr Leu Leu Asp Lys Asp 1 5 9 10 PRT ArtificialSequence Description of Artificial Sequence NHR Sensor Peptides 9 GlnLeu Leu Arg Tyr Leu Leu Asp Lys Asp 1 5 10 10 11 PRT Artificial SequenceDescription of Artificial Sequence NHR Sensor Peptides 10 His Gln LeuLeu Arg Tyr Leu Leu Asp Lys Asp 1 5 10 11 14 PRT Artificial SequenceDescription of Artificial Sequence NHR Sensor Peptides 11 Pro Gln AlaGln Gln Lys Ser Leu Leu Gln Gln Leu Leu Thr 1 5 10 12 8 PRT ArtificialSequence Description of Artificial Sequence NHR Sensor Peptides 12 LeuLeu Gln Gln Leu Leu Thr Glu 1 5 13 9 PRT Artificial Sequence Descriptionof Artificial Sequence NHR Sensor Peptides 13 Val Thr Leu Leu Gln LeuLeu Leu Gly 1 5 14 8 PRT Artificial Sequence Description of ArtificialSequence NHR Sensor Peptides 14 Ile Leu Arg Lys Leu Leu Gln Glu 1 5 15 8PRT Artificial Sequence Description of Artificial Sequence NHR SensorPeptides 15 Ile Leu Lys Arg Leu Leu Gln Glu 1 5 16 8 PRT ArtificialSequence Description of Artificial Sequence NHR Sensor Peptides 16 IleLeu Arg Arg Leu Leu Gln Glu 1 5 17 8 PRT Artificial Sequence Descriptionof Artificial Sequence NHR Sensor Peptides 17 Ile Leu Lys Lys Leu LeuGln Glu 1 5 18 5 PRT Artificial Sequence DOMAIN (1)..(5) Description ofArtificial Sequence NHR Sensor Peptides; first, fourth and fifth residueare independently selected from hydrophobic amino acids; second andthird residues are independently selected from any amino acid. 18 XaaXaa Xaa Xaa Xaa 1 5

What is claimed is:
 1. An in vitro fluorescent polarization assay methodfor characterizing an agent as a ligand of a nuclear hormone receptor,comprising steps: forming an in vitro mixture comprising a purifiednuclear hormone receptor, a sensor and a candidate agent, but not anatural coactivator protein of the receptor, the sensor consisting of apeptide comprising the sequence L₁X₁X₂L₂L₃ (SEQ ID NO: 18) covalentlycoupled to a directly detectable fluorescent label, wherein L₁-L₃ areindependently selected from hydrophobic amino acids and X₁-X₂ areindependently selected from any amino acid and wherein the peptideprovides direct, in vitro ligand-dependent binding to the receptor andis 24 or fewer residues in length, wherein the sensor is at aconcentration of less than about 100 nM.; measuring an assayfluorescence polarization of the sensor as an indication of sensorbinding to the receptor in the presence of the agent; comparing theassay fluorescence polarization to a corresponding control fluorescencepolarization, wherein the control fluorescence polarization provides anindication of sensor binding to the receptor in the absence of theagent, and wherein a greater assay fluorescence polarization thancontrol fluorescence polarization indicates that the agent is a ligandof the receptor.
 2. A method according to claim 1, wherein the peptidecomprises a sequence selected from the group consisting of: KLVQLLTTT(SEQ ID NO:1), ILHRLLQE (SEQ ID NO:2), LLRYLLDK (SEQ ID NO:3), LLRYLLD(SEQ ID NO:4), LRYLLD (SEQ ID NO:5), LLRYLL (SEQ ID NO:6), LRYLL (SEQ IDNO:7), LLRYLLDKD (SEQ ID NO:8), QLLRYLLDKD (SEQ ID NO:9), HQLLRYLLDKD(SEQ ID NO:10), PQAQQKSLLQQLLT (SEQ ID NO:11), LLQQLLTE (SEQ ID NO:12),VTLLQLLLG (SEQ ID NO:13), ILRKLLQE (SEQ ID NO:14, ILKRLLQE (SEQ IDNO:15), ILRRLQE (SEQ ID NO:16) and ILKKLLQE (SEQ ID NO:17).
 3. A methodaccording to claim 1, wherein the peptide consists of a sequenceselected from the group consisting of: KLVQLLTTT (SEQ ID NO:1), ILHRLLQE(SEQ ID NO:2), LLRYLLDK (SEQ ID NO:3), LLRYLLD (SEQ ID NO:4), LRYLLD(SEQ ID NO:5), LLRYLL (SEQ ID NO:6), LRYLL (SEQ ID NO:7), LLRYLLDKD (SEQID NO:8), QLLRYLLDKD (SEQ ID NO:9), HQLLRYLLDKD (SEQ ID NO:10),PQAQQKSLLQQLLT (SEQ ID NO:11), LLQQLLTE (SEQ ID NO:12), VTLLQLLLG (SEQID NO:13), ILRKLLQE (SEQ ID NO:14, ILKRLLQE (SEQ ID NO:15), ILRRLLQE(SEQ ID NO:16) and ILKKLLQE (SEQ ID NO:17).
 4. A method according toclaim 1, wherein the sensor is at a concentration of less than about 10nM.
 5. A method according to claim 1, wherein the peptide is 12 or fewerresidues in length.
 6. A method according to claim 1, wherein thefluorescent label is coupled to the N-terminus of the peptide.
 7. Amethod according to claim 2, wherein the sensor is at a concentration ofless than about 10 nM.
 8. A method according to claim 2, wherein thepeptide is 12 or fewer residues in length.
 9. A method according toclaim 2, wherein the fluorescent label is coupled to the N-terminus ofthe peptide.
 10. A method according to claim 3, wherein the sensor is ata concentration of less than about 10 nM.
 11. A method according toclaim 3, wherein the peptide is 12 or fewer residues in length.
 12. Amethod according to claim 3, wherein the fluorescent label is coupled tothe N-terminus of the peptide.
 13. A method according to claim 4,wherein the peptide is 12 or fewer residues in length.
 14. A methodaccording to claim 4, wherein the fluorescent label is coupled to theN-terminus of the peptide.
 15. A method according to claim 13, whereinthe fluorescent label is coupled to the N-terminus of the peptide.
 16. Amethod according to claim 1, wherein the sensor is at a concentrationless than that of the receptor.
 17. A method according to claim 1,wherein the sensor is at a concentration less than that of the receptor,wherein the sensor is at a concentration of 1-5 nM and the receptor isat concentration of 100-200 nM.
 18. A method according to claim 6,wherein the sensor is at a concentration less than that of the receptor,wherein the sensor is at a concentration of 1-5 nM and the receptor isat concentration of 100-200 nM.